Electrical impedance hematocrit and HbA1c biosensor comprising sample plate and sample apparatus

ABSTRACT

A sampling plate ( 1 ) is provided comprising a sample zone ( 2 ) for receiving a liquid sample, and two drive electrodes ( 3, 4 ) with separate respective electrode terminals spaced by a spacing for receiving a the liquid sample within the sample zone for use in driving an electrical signal through the sample. Two sensing electrodes ( 5, 6 ) are provided with separate respective electrode terminals spaced between the electrode terminals of the two drive electrodes for use in sensing an electrical signal generated by the drive electrodes within a the sample. A sampling apparatus ( 15 ) is provided for use with the plate.

RELATED APPLICATIONS

This application is divisional of U.S. application Ser. No. 14/394,176,filed Oct. 13, 2014, which in turn is a 371 application of InternationalApplication No. PCT/GB2013/050957 filed Apr. 12, 2013, which claims thebenefit of priority of United Kingdom Patent Application No. 1206588.4filed Apr. 13, 2012. Each of the foregoing applications is herebyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a sample measurement system. Inparticular, the present invention relates to the measurement ofproperties of liquid samples of (or containing) blood. In particular theinvention relates to a sample measurement system for measuring certainselected properties of a liquid substrate, such as the glucose levels ina blood sample. The invention also relates to a sampling plate, ameasurement device, a data carrier containing software to operate themeasurement device.

BACKGROUND OF THE INVENTION

There is a widespread need for improving the accuracy of samplemeasurement systems such as those enabling e.g. a diabetes sufferer toknow their blood sugar levels—i.e. the concentration of glucose in theirblood.

Existing sample measurement systems use a measurement device whichreceives and takes measurement readings from a sampling plate spottedwith a blood sample from a user. The sampling plate is often rectangularand is end-loaded with the blood sample. The blood sample, once loaded,is usually drawn into a sample zone having a number of sampling zonesfrom which measurements are taken by the system.

Each sampling zone typically has its own particular contents. Forexample, the first sampling zone may have a glucose oxidase depositwithin it, a second deposit comprising a mixture of glucose oxidase anda predetermined amount of glucose, while a third sampling zone maycontain no deposit. As the blood sample is drawn over all three samplingzones, chemical reactions occur with the deposits in each sampling zone,resulting in discrete electrolytes. Each sampling zone bridges acorresponding pair of electrodes. A potential difference is establishedacross each sampling zone, via the electrodes, when the sampling plateis inserted into an operating measurement device. Electric currentreadings for each sampling zone then provide measurements necessary toassess the blood sugar (glucose) levels. For instance, the firstsampling zone may give the primary measurement, whereas the secondsampling zone may provide a degree of calibration since a known quantityof glucose was already present there. The third zone may give a finalcheck by accounting for the non-glucose contribution to the measurementsin the first and second sampling zones.

However, in spite of these calibrations and final checks, error marginsin such blood glucose readings are still high. Indeed, blood glucoselevels are strongly influenced by the fluctuating and transient glucoselevels in the plasma of the blood sample, which may not berepresentative of the long-term blood glucose levels of the patient andmay, rather, simply indicate a recent transient rise or drop in bloodglucose levels within the blood plasma of the patient e.g. due to recentfood consumption of other short-term environmental factors.

The present invention aims to address this.

Blood plasma is the liquid component of blood in which the blood cellsin whole blood are normally suspended. Blood plasma typicallyconstitutes about 55% of the total volume of the blood. It is theextracellular fluid part of blood and is mostly water but containsdissolved glucose and other contents.

The volume percentage of red blood cells in blood is known as thehaematocrit (HCT). Other terms for this are the packed cell volume (PCV)or erythrocyte volume fraction (EVF). Haematocrit is normally about 45%for men and 40% for women. The haematocrit is typically calculated bymultiplying the red blood cell count in a blood sample by the averagecell volume, then dividing the result by the whole blood sample volume.

Glycated haemoglobin (a.k.a. haemoglobin A1c, HbA1c, or just A1c) is aform of haemoglobin measured primarily to identify the average plasmaglucose concentration over prolonged periods of time. It is formed in anon-enzymatic glycation pathway by hemoglobin's exposure to plasmaglucose. Normal levels of glucose produce a normal amount of glycatedhemoglobin. As the average amount of plasma glucose increases, thefraction of glycated hemoglobin increases. This serves as a marker foraverage blood glucose levels over the previous months prior to themeasurement. Liquid chromatography and capillary electrophoresis are twoways of measuring glycated haemoglobin (HbA1c). Both methods arecomplex, expensive and wholly unsuited for easy and simpleimplementation by a patient.

BRIEF SUMMARY OF THE INVENTION

At its most general, the invention in one aspect is a system (methodand/or apparatus) to measure haematocrit of a liquid sample containingblood according to the electrical impedance (e.g., resistance andreactance) it has in response to an alternating electrical potentialdifference applied across the sample. The measured haematocrit may beused to improve the accuracy of blood glucose measurements of the bloodin another aspect of the invention. It has been found that applying analternating potential difference (voltage) across such a sample resultsin a resistance and/or a reactance which is surprisingly responsive tohaematocrit. The invention exploits this finding. The presence of redblood cells within a blood sample complicates the interpretation ofblood glucose measurements using existing methods. The invention mayremove or reduce that complication to enable more accurate blood glucosemeasurements to be made. At its most general, the invention in anotheraspect is a system (method and/or apparatus) to measure a level ofglycated haemoglobin (HbA1c) in a liquid sample containing bloodaccording to the electrical impedance (e.g., resistance and reactance)it has in response to an alternating electrical potential differenceapplied across the sample. It has been found that applying analternating potential difference (voltage) across such a sample resultsin a resistance and/or a reactance which is surprisingly responsive toHbA1c of the sample. The invention exploits this finding.

In a first of its aspects, the invention may provide a sampling platecomprising a sample zone for receiving a liquid sample. The samplingplate may have two drive electrodes with separate respective electrodeterminals spaced by a spacing for receiving the liquid sample within thesample zone for use in driving an electrical signal through the sample.Two sensing electrodes may be provided with separate respectiveelectrode terminals spaced between the electrode terminals of the twodrive electrodes for use in sensing an electrical signal generated bythe drive electrodes within a the sample.

Herein, a “sampling plate” may mean any surface capable of receiving aliquid sample in a sample zone. Preferably, however, the sampling plateis portable. Suitably the sampling plate may cover an area less than 1m², preferably less than 50 cm², more preferably less than 10 cm² andmost preferably less than 5 cm². The sampling plate may cover an arealess than 500 mm²—for instance 350 mm² where the sampling plate is 10 mmwide by 35 mm long. Suitably the sampling plate may be rectangular. Thesampling plate may be a strip, and may be a flexible strip. Preferably,however, the sampling plate is an individual plate, preferably a rigidsampling plate. The thickness of the sampling plate is preferably lessthan 1 cm, preferably less than 1 mm, more preferably less than 0.5 mm,most preferably less than 0.25 mm.

The sampling plate is preferably compatible with a measurement device.For example, the measurement device is preferably operable tocommunicate with the sampling plate to measure one or more selectedproperties of the sample. Preferably the sampling plate may be insertedinto the measurement device to allow measurements to be taken.

The two sensing electrode terminals may present to each other opposingsides which define between them an elongate sensing gap extending alongthe sample zone for receiving at least parts of the sample therein. Thesensing electrode terminals may be substantially flat and side-by-sideto define a substantially flat sensing gap. The width of the two sensingelectrode terminals is preferably the same. That width is preferablyabout double the size of the sensing gap between them.

The two drive electrode terminals may present to each other opposingsides which define between them an elongate drive gap extending alongthe sample zone for receiving at least parts of the sample thereinwhereby the drive electrodes are adapted to drive electrical signaltransversely across the drive gap. The drive electrode terminals may besubstantially flat and side-by-side to define a substantially flat drivegap.

The sensing gap may extend along the drive gap. The sensing gap and/orthe drive gap may preferably have a substantially uniform width along atleast a part of its length.

A drive electrode terminal and an adjacent sensing electrode terminalmay be arranged in/on the sampling plate so that they present to eachother opposing sides which define between them an elongate partitioninggap. Preferably, this partitioning gap extends along the sample zone todefine a partition between those adjacent terminals within the samplezone. This may apply to each of the drive electrode terminals and theirrespective adjacent/neighbouring sensing electrode terminal.

The partitioning gap preferably has a substantially uniform width alongat least a part of its length, preferably substantially all of itslength. Each partitioning gap width is preferably the same size.Preferably the partitioning gap width is about 1% times the width of thesensing gap.

The sensing electrode terminals may be formed on a surface of thesensing plate within the sample zone. Preferably, the drive electrodeterminals are formed on a surface of the sensing plate in common withthe sensing electrode terminals within the sensing zone. The electrodesmay be formed upon the sampling plate by a known printing process.However, for a better degree of accuracy or consistency a techniqueknown as laser ablating is preferably used to remove electrode material(e.g. Gold) formed as a sheet/coating onto a surface of the samplingplate, where the electrode gaps are required. Preferably, in production,conductive electrode material may be laid down on a surface area on oneside of the sampling plate with no gap features, and the gaps may thenbe laser ablated to define the electrodes.

The sampling plate is preferably arranged to be detachably attachableelectrically to electrical apparatus adapted for supplying the drivecurrent to the drive electrodes and for taking measurements via thesensing electrodes. In this regard, preferably each of the driveelectrodes and each of the sensing electrodes is in electricalcommunication with respective electrical contacts provided on thesampling plate which are exposed for electrical connectionsimultaneously with an external drive current source and externalsensing circuitry, respectively, of such an apparatus.

Preferably, the width of the sensing gap is greater than about 90microns and less than about 160 microns. Preferably the width of thepartitioning gap is about 1.5 times the width of the sensing gap. Analternating potential difference is preferably applied across a gapbetween two electrodes designed to be bridged by a sample beingmeasured. It has been found that a careful dimensioning of the sensinggap and the drive gap enhances the accuracy of haematocrit measurementgreatly, and also permits HbA1c to be measured. The gap size ispreferably substantially smaller than a gap size between electrodestypically employed in existing systems designed to measure bloodglucose. Preferably, the sensing gap is wider than the average width ofa human red blood cell, but less than the average width of two suchcells.

It is postulated, but not asserted, that the sensing gap serves to forma generally linear array of red blood cells along it in which the arrayis generally one cell in width—this being constrained by the width ofthe sensing gap—and that the parts of the sensing gap not occupied byred blood cells are occupied by blood plasma. Blood plasma is typicallymore electrically conductive than are red blood cells at certainelectrical signal frequencies. By applying an oscillating voltage, redblood cells remain mobile (e.g. oscillate) within the gap and may notcoat one or other of the electrodes. The result may be that there ismaintained within the sensing gap a defined linear array of red bloodcells mobile within conductive blood plasma. The proportion of red bloodcells within the gap, relative to the quantity of blood plasma there,influences the quantity of electrically conductive pathways (throughplasma, around blood cells) available to currents applied. This maymanifest itself as an electrical impedance value (e.g. resistance,reactance) determined by a haematocrit value and/or an HbA1c value, ashas been observed.

The sample zone may comprise a reagent to react with free glucose in theliquid sample. This may be so when the sampling plate is intended foruse in measuring a value representing the concentration of glycatedhaemoglobin (HbA1c) in the liquid sample as described below. The reagentmay be a deposit formed on one (e.g. exclusively) of the driveelectrodes in the sample zone (e.g. the one for use as an anode) to bedirectly accessible to a sample therein. The deposit may be in the formof an ink or paste. Preferred reagents are oxidising agents. Mostpreferred are enzymes and especially preferred are glucose oxidase (GOx)and glucose dehydrogenase (GDH). Where no such reagent is present, thesampling plate may be used for measuring haematocrit as described below.

The sampling plate may comprise a further sample zone containing a pairof drive electrodes as described above, and a pair of sensing electrodesas described above. The further sample zone may be free of any reagentand be intended for use in measuring HCT of a blood sample, while theother sample zone may contain the reagent and be intended for concurrentor sequential measurement of HbA1c of the same sample.

Alternatively, the drive electrode terminals of the further sample zonemay comprise only one pair of drive electrodes which may present to eachother, across a respective spacing, opposing electrode sides extendingalong the sample zone. The further sample zone may contain the reagentand be intended for use in measuring blood glucose levels within theplasma of a blood sample. These opposing sides may define between them adrive gap for receiving a sample therein. This spacing may define a gapwhich is preferably greater than about 200 microns in width, and may bebetween 200 microns and 400 microns in width. This dimensioning has beenfound to be preferable for the electrodes in sampling zones containingthe reagent to react with free glucose in the liquid sample, and formeasuring a current generated in response to a direct (DC) drive voltageapplied across the drive gap. The measured current may be used todetermine a measure of the glucose in the blood plasma of the sample.

The opposing sides in the pair of drive electrodes of the further samplezone may be of unequal length. They may be curved. One side may beconvex and the opposing side reciprocally concave and of greater lengththan the convex side. Preferably the electrode with the longer side isused as the cathode of the pair. This is preferable in view of thegreater gap size in each further pair of electrodes. It has been foundthat electrical currents driven across those wider gaps through a bloodsample are more prone to diffuse in a direction along the gap ratherthan flowing directly across the gap un-deviated. In order to bettercapture diffused charges (current) in the blood sample the electrode towhich the charges flow when a direct (DC) voltage is applied between theelectrodes, has the longer edge. The spacing of the drive gap may besubstantially uniform along at least a part of its length.

In a second of its aspects, the invention may provide a samplingapparatus for use in performing electrical measurements on a liquidsample containing blood, including two current output terminals foroutputting an alternating current signal applied therebetween, and analternating electrical current unit in electrical communication with thetwo current output terminals for applying thereto an alternatingelectrical current of a given amplitude and frequency when a liquidsample is in electrical connection between the two current outputterminals.

The sampling apparatus may include a voltage unit in electricalcommunication with the two current output terminals for applyingtherebetween a direct (DC), being most preferably a substantiallyconstant (DC), electrical potential difference of a given magnitude. Afirst voltage input terminal may be provided for receiving a firstelectrical signal externally input thereto and a separate second voltageinput terminal for receiving a second electrical signal externally inputthereto when the liquid sample is in electrical connection between thefirst and second voltage input terminals. The apparatus may includevoltage detector(s) for measuring a first voltage and a second voltageusing said first and second electrical signals, respectively.

A control unit may be arranged in the sampling apparatus to control theelectrical current unit to apply the alternating electrical current ofgiven frequency and concurrently to control the voltage unit and thevoltage detector(s) to measure the first and second voltages both whenthe direct (e.g. substantially constant) DC electrical potentialdifference is applied and when the direct DC electrical potentialdifference is not applied.

A calculating unit may be arranged in the sampling apparatus tocalculate a first electrical impedance (e.g. reactance) value using thefirst and second voltages measured when the direct (e.g. substantiallyconstant) DC electrical potential difference is applied, and tocalculate a second electrical impedance (e.g. reactance) value measuredwhen the direct DC electrical potential difference is not applied.

The calculating unit may be arranged in the sampling apparatus togenerate a value representing the concentration of glycated haemoglobin(HbA1c) in the liquid sample according to the first electrical reactancevalue, the second electrical reactance value and a value representingthe relative volume of red blood cells in the liquid sample(haematocrit, HCT).

The calculating unit may be arranged to generate a value representingthe concentration of glycated haemoglobin (HbA1c) in the blood withinthe sample according to the first electrical reactance value, the secondelectrical reactance value and a value of the relative volume of redblood cells in the liquid sample (haematocrit, HCT) according to thefollowing formula, and store the result and/or to output the result tothe user:

${{HbA1c} = {100 \times \left( {1 - \frac{X_{1}}{HCT \times X_{2}}} \right)}}.$

The haematocrit value HCT may be a contemporaneously measured value,such as measured using a sample of the blood on the sampling plate, ormay be a predetermined value which is generated independently of thesampling unit.

The quantity X₁ is considered to represent the reactance of a bloodsample due to glycated red blood cells in the blood within the firstsampling zone from which free glucose has been substantially oxidized bythe reagent, whereas X₂ is considered to represent the reactance of thewhole blood sample in which both plasma and red blood cells containglucose. The proportion of that reactance due to red blood cells isconsidered to be represented by the term (HCT)×(X₂) according to thehaematocrit of the sample.

The given frequency preferably has a value in the range 500 KHz to 1.5MHz, e.g. about 1 MHz. More preferably, the given frequency has a valuein the range 750 KHz to 1.25 MHz, yet more preferably the givenfrequency has a value in the range 850 KHz to 1.15 MHz, even morepreferably the given frequency has a value in the range 900 KHz to 1.1MHz, yet even more the given frequency has a value in the range 970 KHzto 1.03 MHz. It has been found that a frequency of about 1 MHz worksespecially well, and frequencies reasonably close to this value aredesirable, though the ranges given above have been found to beacceptable in terms of accuracy of measurement in implementing theinvention. The value of the direct (DC) voltage may be a value in therange from about 0.01 volts to about 1.0 volts, or preferably from about0.1 volts to about 0.5 volts, or more preferably from about 0.2 volts toabout 0.3 volts—e.g. about 0.25 volts.

It is postulated, but not asserted, that the presence of a direct (DC)voltage across the drive gap, and therefore across the sensing gap, hasthe effect of polarizing or physically aligning in a common directionthose red blood cells that are not glycated, while the glycated redblood cells are not forced into this alignment and remain largelyunaffected by the direct voltage applied across the sample. Theconsequence is felt most keenly when an alternating (AC) current isapplied to the blood sample while the is concurrently subjected to thisDC voltage. The result is believed to be that the un-glycated red bloodcells aligned by the applied DC voltage are far less responsive to theAC current concurrently applied (i.e. less able to dynamicallyinteract/oscillate in response to it) than are the glycated red bloodcells. The result is that the portion of the impedance (e.g. reactance)of the blood sample arising from the un-glycated red blood cells fallsdramatically, leaving the glycated red blood cells to dominate theimpedance of the sample. By comparing this impedance value to theimpedance value of the same sample measured when no direct (DC) voltageis applied (and thus, no un-glycated cell alignment occurs) provides aroute to determining the proportion of glycated red blood cells in thesample and, from that, a measurement of HbA1c.

Alternatively, or additionally, the calculating unit may be arranged togenerate a value representing the relative volume of red blood cells inthe liquid sample (haematocrit) according to electrical impedance (e.g.resistance and reactance) values measured thereby from the sample. Thecalculating unit may be arranged to generate a value representing theconcentration of glycated haemoglobin in the liquid sample accordingthereto.

In a third aspect, the invention may provide a sampling apparatus foruse in performing electrical measurements on a liquid sample containingblood, the apparatus comprising two current output terminals foroutputting an alternating current signal applied therebetween, and analternating electrical current unit in electrical communication with thetwo current output terminals for applying therebetween an alternatingelectrical current of a given amplitude and frequency, when a liquidsample is in electrical connection between the two current outputterminals.

This sampling apparatus may include a first voltage input terminal forreceiving a first electrical signal externally input thereto and aseparate second voltage input terminal for receiving a second electricalsignal externally input thereto, when said liquid sample is inelectrical connection between the first and second voltage inputterminals, and a voltage detector(s) for measuring a first voltage and asecond voltage using said first and second electrical signals,respectively.

A control unit may be arranged in this sampling apparatus to control theelectrical current unit to apply the alternating electrical current at afirst frequency and concurrently to control the voltage detector(s) tomeasure the first and second voltages, and to further control theelectrical current unit to apply the alternating electrical current at asecond frequency exceeding the first frequency and concurrently tocontrol the voltage detector(s) to measure the first and secondvoltages. The first frequency may be a value (e.g. 50 KHz) within afirst continuous range of values from about 1 KHz to about 150 KHz. Morepreferably, the first frequency has a value in the range 25 KHz to 125KHz, yet more preferably the first frequency has a value in the range 35KHz to 100 KHz, even more preferably the first frequency has a value inthe range 45 KHz to 75 KHz, yet even more the first frequency has avalue in the range 47 KHz to 53 KHz. It has been found that a frequencyof about 50 KHz works especially well, and frequencies reasonably closeto this value are desirable, though the ranges given above have beenfound to be acceptable in terms of accuracy of measurement inimplementing the invention.

The second frequency may be a value (e.g. 1 MHz) within a secondcontinuous range of values from about 500 KHz to about 1.5 MHz. Morepreferably, the second frequency has a value in the range 750 KHz to1.25 MHz, yet more preferably the second frequency has a value in therange 850 KHz to 1.15 MHz, even more preferably the second frequency hasa value in the range 900 KHz to 1.1 MHz, yet even more the secondfrequency has a value in the range 970 KHz to 1.03 MHz. It has beenfound that a second frequency of about 1 MHz works especially well, andfrequencies reasonably close to this value are desirable, though theranges given above have been found to be acceptable in terms of accuracyof measurement in implementing the invention.

In general, the preferred range of frequencies, and the preferentialfrequency within such a range, is influenced to some extent bygeometrical considerations of the sampling process. Factors such as thesize of surface area of conductive elements/electrodes within a testarea of a sampling plate, in relation to the size of surface area ofnon-conductive/non-electrode parts between electrodes, can influence theposition and extent of the suitable AC signal frequency ranges. Thesesurface areas may typically be located within a sampling area, well orzone within a sampling plate which is between about 0.5 mm and 5 mm indiameter or width, or more preferably between about 1 mm and 3 mm, suchas about 1.6 mm in diameter or width. These dimensions enable a samplesize which is large enough to do reliable measurement upon, but does notresult in a sampling size (or sampling plate size) which is too largefor these purposes, or for practical use generally.

This sampling apparatus may include a calculating unit arranged tocalculate a first electrical impedance (e.g. resistance and/orreactance) value using the first and second voltages measured at thefirst frequency, and a second electrical impedance (e.g. resistanceand/or reactance) value and a reactance value using the first and secondvoltages measured at the second frequency. The first electricalimpedance may be a resistance value (R₁, ohms). The second impedance maybe comprise both a resistive part (R₂, ohms) and a reactive part (X₃,ohms). The calculating unit may be arranged to generate a valuerepresenting the relative volume of red blood cells in the liquid sample(haematocrit, HCT) according to the first and second electricalresistance values (R₁, R₂) and the electrical reactance value (X₃).

The sampling apparatus may be arranged to calculate HCT according to thefollowing equation:

${HCT} = {\left\lbrack {{A{\ln\left( \frac{R_{1}}{R_{2}} \right)}} + {B{\ln\left( {X_{3} + X_{0}} \right)}} - C} \right\rbrack.}$

The quantities A, B and C are preferably constants associated with asampling plate design in use. For example, the values of A, B and C mayeach typically be within the range from about 0.05 to about 0.5, orpreferably between about 0.1 and 0.25, or more preferably between about0.1 and about 0.2. For example, the electrodes of the sampling sheet maybe formed from a conductive material (e.g. a metal such as Gold) havinga sheet resistance of 5 ohms per square, the values in question may be:A=0.142; B=0.155; C=0.157. The value of the term A has been found to beaffected by the electrical properties of the electrodes of the samplingplate (e.g. drive electrode terminals and/or sensing electrodeterminals) within the sampling zone(s). Different properties such asconductivity (e.g. sheet resistance), the electrical voltages andcurrents applied to the electrodes in the sampling zone(s), and thegeometry (e.g. widths) of the drive electrode terminals and sensingelectrode terminals. The value of the term B has been found to beaffected by the nature of the interaction and interface between theblood sample and the sampling strip surface in the sampling zone. Forexample, the microscopic surface roughness and the “wetting ability” ofthe surface affect the value of this term. Also, the aspect ratio of theelectrodes within the sampling zones (e.g. the blood sample “height” ascompared to the area of the electrode surfaces in the sampling zone overwhich it is arranged) can affect the value of B—thus, thethree-dimensional geometry (e.g. depth) of the sampling zone plays arole. The term C has been found to be affected by the geometry of theshape of the sample shape determined by the shape of the sampling zone,in a way similar to its influence on the term B. The electrodes may havea sheet resistance in the range from about 2 ohms per square to about 15ohms per square.

Actual values, suited to a given sampling zone geometry and electrodestructure and material, may be determined by routine calibrationemploying commercially available blood samples of known HCT, as will beapparent to the skilled person. The value of X₀ may simply be zero, ormay be adjusted if necessary to improve the predictive accuracy of theequation.

The sampling apparatus may be arranged to generate both the valuerepresenting the relative volume of red blood cells in the liquid sample(haematocrit) as described above, and to generate the value representingthe concentration of glycated haemoglobin (HbA1c) in a liquid sample asdescribed above, using that haematocrit.

The sampling apparatus may include the sampling plate described above.For example, each one of the two drive electrodes of the sampling platemay be adapted to electrically connect to a respective one of the twocurrent output terminals concurrently. Furthermore, each one of the twosensing electrodes of the plate may be adapted to electrically connectto a respective one of the first voltage input terminal and the secondvoltage input terminal concurrently, thereby to connect the two driveelectrodes and the two sensing electrodes to the sampling apparatussimultaneously for electrical communication therewith.

In another of its aspects, the invention may provide sampling apparatus(e.g. measurement device) for use in performing electrical measurementson a liquid sample containing blood, the apparatus comprising: a firstoutput terminal arranged for outputting an alternating (AC) electricalcurrent; and a second output terminal arranged outputting a directelectrical voltage applied thereto (most preferably a substantiallyconstant (DC) voltage); and voltage input terminals (e.g. two) each forreceiving an input electrical voltage signal externally input thereto;and current input terminals (e.g. two) each for receiving an inputelectrical current signal externally input thereto. The apparatus mayinclude a control unit arranged to apply an alternating electricalcurrent to the first output terminal and concurrently to measure a firstelectrical voltage at the voltage input terminals resulting therefromwhen a the liquid sample is in electrical series connection between thefirst output terminal and a current input terminal, and arranged toapply a direct voltage (most preferably a substantially constantelectrical (DC) voltage) to the second output terminal and concurrentlyto measure a second electrical current at a current input terminalresulting therefrom when a liquid sample is in electrical seriesconnection between the second output terminal and a current inputterminal. A calculating unit of the apparatus may be arranged tocalculate electrical resistance and/or reactance values for the sampleusing a value of the first electrical current and a value of theconcurrently measured first voltage, and arranged to calculate a firstcalculated value representing the relative volume of red blood cells inthe liquid sample (haematocrit) according to the calculated electricalresistance and/or reactance values; and to calculate a second calculatedvalue representing an amount of glucose in the liquid sample accordingto both the first calculated value and the measured second electricalcurrent, and to output the result. The measured second electricalcurrent may be measured while the direct (DC) voltage is applied to asample zone of a sampling plate that contains a deposit of reagent toreact with free glucose in a blood sample when applied there for use inmeasuring a first value for blood glucose levels within a blood samplewhen there. The alternating current may be applied to a sample zone freeof such reagent and for use in measuring haematocrit within a bloodsample when there. The haematocrit value may be used to improve thefirst value for blood glucose levels within a blood sample.

In a fourth of its aspects, the invention may provide sample measurementmethod for performing electrical measurements on a liquid samplecontaining blood, the method comprising receiving the liquid sample on asample plate comprising electrode terminals which are separated by aspacing adapted to be bridged by blood from the liquid sample and whichcomprise a reagent to react with free glucose in the liquid sample, andapplying to the electrodes an alternating electrical current having agiven frequency to generate a first alternating potential differenceacross the spacing between the electrode terminals. The method mayinclude also applying between the electrode terminals a substantiallyconstant (DC) electrical potential difference of a given magnitude, anddetermining a value of a first electrical reactance of the liquid samplebridging said spacing for said given frequency, then removing thesubstantially constant (DC) electrical potential difference from betweenthe two electrode terminals. The method may include applying to theelectrodes the alternating electrical current having the givenfrequency, without the DC potential applied, to generate a secondalternating potential difference across the spacing between theelectrode terminals, and determining a value of a second electricalreactance of the liquid sample bridging the spacing for the givenfrequency. The method may include generating a value representing theconcentration of glycated haemoglobin (HbA1c) in the blood within thesample according to the first electrical reactance value, the secondelectrical reactance value and a value of the relative volume of redblood cells in the liquid sample (haematocrit). The given frequencypreferably has a value in the range 500 KHz to 1.5 MHz, e.g. about 1MHz. More preferably, the given frequency has a value in the range 750KHz to 1.25 MHz, yet more preferably the given frequency has a value inthe range 850 KHz to 1.15 MHz, even more preferably the given frequencyhas a value in the range 900 KHz to 1.1 MHz, yet even more the givenfrequency has a value in the range 970 KHz to 1.03 MHz.

In a fifth aspect, the invention may provide a sample measurement methodfor performing electrical measurements on a liquid sample containingblood, the method comprising receiving the liquid sample on a sampleplate comprising electrode terminals which are separated by a spacingadapted to be bridged by blood from the liquid sample, and applying tothe electrodes an alternating electrical current having a first signalfrequency to generate a first alternating potential difference acrossthe spacing between the electrode terminals. This method may includedetermining a value of a first electrical resistance of the liquidsample bridging the spacing for the first signal frequency. The methodmay include applying to the electrodes an alternating electrical currenthaving a second signal frequency exceeding the first signal frequency togenerate a second alternating potential difference across the spacingbetween the electrode terminals, and determining a value of a secondelectrical resistance and a value of a reactance of the liquid samplebridging the spacing for the second signal frequency. This method mayinclude generating a value for the relative volume of red blood cells(haematocrit) in the liquid sample according to the first electricalimpedance value and the second electrical impedance value. The firstfrequency preferably has a value in the range 1 KHz to 150 KHz, e.g.about 50 KHz. More preferably, the first frequency has a value in therange 25 KHz to 125 KHz, yet more preferably the first frequency has avalue in the range 35 KHz to 100 KHz, even more preferably the firstfrequency has a value in the range 45 KHz to 75 KHz, yet even more thefirst frequency has a value in the range 47 KHz to 53 KHz. The secondfrequency preferably has a value in the range 500 KHz to 1.5 MHz, e.g.about 1 MHz. More preferably, the second frequency has a value in therange 750 KHz to 1.25 MHz, yet more preferably the second frequency hasa value in the range 850 KHz to 1.15 MHz, even more preferably thesecond frequency has a value in the range 900 KHz to 1.1 MHz, yet evenmore the second frequency has a value in the range 970 KHz to 1.03 MHz.

The invention in its fourth aspect may comprise generating a valuerepresenting the concentration of glycated haemoglobin (HbA1c) in theblood within the sample using the value representing the relative volumeof red blood cells in the liquid sample (haematocrit) as generatedaccording to the invention in its fifth aspect.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

To better illustrate the invention there now follows a non-limitingexamples of embodiments of the invention with reference to theaccompanying drawings of which:

FIG. 1 illustrates schematically a sampling plate attached to a samplingunit;

FIG. 2 illustrates the electrode terminals of the sampling plate in moredetail;

FIG. 3 illustrates the sampling plate in isolation, in the form of adisposable sampling strip;

FIG. 4 illustrates schematically a sampling plate and sampling apparatusaccording to another embodiment of the invention, comprising two samplezones and electrode groupings;

FIG. 5 schematically illustrates equivalent circuit diagrams;

FIG. 6 schematically shows a sampling plate and sampling unit containingan ASIC comprising circuitry components adapted to implement signalgeneration and reception to and from a sampling plate;

FIG. 7 schematically shows a sampling plate and sampling unit containingan ASIC comprising circuitry components adapted to implement signalgeneration and reception to and from a sampling plate.

DETAILED DESCRIPTION OF THE INVENTION

In the drawings, like items are assigned like reference symbols.

FIG. 1 shows a sampling plate (1) in the form of a strip of firm andnon-conductive material (e.g. plastic) possessing a circular sample zone(2) defined by a circular recess formed within the strip for receiving aliquid blood sample. Within the sample zone there are four electrodeterminals (3, 4, 5, 6) formed upon a surface of the plate forming thefloor of the sample zone and exposed for contact with a received sample.The electrode terminals each comprise a layer of inert conductivematerial, preferably Gold.

The four electrode terminals comprise two drive electrode terminals (3,4) each of which is in the shape of a circular segment the curved edgeof which coincides with a part of the circular edge of the circularsample zone. The straight segment edge of each one of the two driveelectrode terminals is parallel to and opposes the straight segment edgeof the other of the two drive electrode terminals to define between thema straight, elongate drive gap of uniform width within the sample zoneacross which the drive electrode terminals oppose each other and acrosswhich a drive current is driven as explained in more detail below.

Within the elongate drive gap extend two straight parallel sensingelectrode terminals (5, 6) in the form of two strips separated from eachother by a sensing gap of uniform width defined by the spacing betweenopposing side edges of each strip. The opposing edges of the two sensingelectrodes are substantially parallel to each straight segment edge ofeach of the two drive electrode terminals.

Furthermore, the straight segment edge of each one of the two driveelectrode terminals (3, 4) opposes a correspondingly straight andparallel edge of an adjacent sensing electrode terminal (6, 5) to definetherebetween a substantially straight and elongate partitioning gapwhich extends along the sample zone in parallel to the sensing gap.Thus, the two sensing electrodes define a straight and uniform sensinggap of receiving parts of the blood sample within the sensing zone, andthe two drive electrode terminals define, together with neighbouringsensing electrode terminals, two partitioning gaps either side of thesending electrodes which are parallel to each other and to the sensinggap, and which separate the sensing electrode terminals from the driveelectrode terminals within the sensing zone.

The width of the sensing gap is preferably between about 90 microns andabout 150 microns, for example about 100 microns and is dimensioned toadmit, at any point along the sensing gap, a single human blood cellwithout permitting that blood cell to bridge the gap and concurrentlycontact both of the two sensing electrodes defining the sensing gap.Rather, the gap is dimensioned to allow a blood cell space to oscillatewithin the gap between the opposing sensing electrodes in response to analternating current driven transversely across the sensing gap betweenthe two drive electrode terminals (3, 4). In this way, a row of bloodcells may be arranged along the sensing gap when a liquid blood sampleis received within the sensing zone and may be subject to an alternatingdrive current directed transversely (e.g. substantially perpendicular)to the row of cells.

This geometry, and the linear array of single blood cells it enables inuse, has been found to provide a surprisingly accurate and stable meansof measuring not only haematocrit (HCT) values for the blood sample, butalso has been found to enable accurate measurement of the concentrationof glycated haemoglobin in the blood sample (i.e. the so-called “fixed”glucose level within blood cells, or the so-called HbA1c value).Accurate determination of the former has been found to be important forenabling accurate determination of the latter—i.e. one needs to know howmuch of the sample is comprised of red blood cells in order to be ableto determine the quantity of fixed glucose they carry. The presentinvention could be implemented to do both simultaneously or sequentiallywith reliability and accuracy.

The width of each of the two parallel partitioning gaps, either side ofthe sensing electrode terminals is preferably about 1.5 times the valueof the width of the sensing gap. Again, this gap dimension and theparallel arrangement of blood cells the partitioning gaps enable, hasbeen found to assist in providing accuracy and stability.

Each of the electrode terminals within the sensing zone is electricallyconnected to a respective electrical conductor line formed within thebody of the sensing plate so as to be electrically insulated along itslength until terminating at an exposed electrical contact zone at an endor side of the sampling plate distal from the sample zone. For example,the first drive electrode terminal (3) is electrically connected to afirst drive contact zone (14) via a first (10) electrical conductorstrip (e.g. Gold). The first sensing electrode terminal (6) iselectrically connected to a first sensing contact zone (13) via a second(9) electrical conductor strip (e.g. Gold). Similarly, the second sensorelectrode terminal (5) is electrically connected to a second sensingcontact zone (12) via a third (8) electrical conductor strip (e.g.Gold). Finally, the second drive electrode terminal (4) is electricallyconnected to a second drive contact zone (11) via a fourth (7)electrical conductor strip (e.g. Gold).

These four contact zones are arrayed in a line along an edge of thesensing plate, at the distal end of the strip, to permit the end of thestrip to be inserted into an electrical socket/port of an electricalsensing unit (15) to place the each one of the four contact zonessimultaneously in electrical connection with a respective one of four(16, 17, 19, 20) electrical contact terminals of the sensing unit.

The sensing unit may be a handset, or part of a larger piece ofequipment. The sensing unit comprises an alternating current source (18)arranged to generate an alternating electrical current of selectedamplitude and selected frequency, and apply the alternating current to afirst and second sensing contact terminals (16, 17) for application tothe drive electrode terminals (3, 4) as a drive current via the firstand second drive contact zones of the sensing plate. A control processorunit (23) is operatively connected to the current source (18) to controlthe frequency of the generated current signal. For example, the controlprocessor may control the current signal frequency to be a value (e.g.50 KHz) within a first continuous range of values from about 1 KHz toabout 150 KHz, or to be a value (e.g. 1 MHz) within a second continuousrange of values from about 500 KHz to about 1.5 MHz. The controlprocessor is arranged to selectively switch the frequency value from afirst value within the first range to a second value within the secondrange.

A detector unit (21) is electrically connected to a first and secondcontact terminals (19, 20) for receiving voltage signals from the firstand second sensing electrodes (6, 5) via the first and second sensingcontact zones of the sensing plate.

The control processor (23) is arranged to control the electrical currentgenerator to apply an alternating electrical current at a firstfrequency (selected from within the first range of values) andconcurrently to control the voltage detector (21) to measure a firstvoltage signal received via the first sensing electrode and to measure asecond voltage signal received via the second sensing electrode.Subsequently, the control processor further controls the electricalcurrent generator to apply an alternating electrical current at a secondfrequency exceeding the first frequency (selected from the secondfrequency range) and concurrently to control the voltage detector (21)to measure a third and a fourth voltage signal value received from thefirst and second sensing electrodes respectively.

The control processor is arranged to calculate a first electricalresistance value (R₁, Ohms) using an amplitude of the voltage differencebetween the first and second voltages measured at the first frequencyand the amplitude of the associated applied alternating current, and tocalculate an electrical impedance value (Z₂=R₂±jX₃, Ohms) using anamplitude of the voltage difference between the third and fourthvoltages measured at the second frequency, and the amplitude of theassociated applied alternating current. Here R represents a resistivecomponent of impedance and X represents a reactive component ofimpedance. The quantity j=√{square root over (−1)}.

Using these two impedance values, the control processor is arranged togenerate a value (HCT) representing the relative volume of red bloodcells in the liquid sample (haematocrit) according to the followingequation.

${HCT} = \left\lbrack {{A{\ln\left( \frac{R_{1}}{R_{2}} \right)}} + {B{\ln\left( {X_{3} + X_{0}} \right)}} - C} \right\rbrack$

This equation is discussed in more detail below (Equation (3)). Thevalues of the constants A, B, C and X₀ may be determined for a givensensing gap and/or partitioning gap width, and electrodestructure/material for a given sensing plate, by routine calibration andexperimentation as would be readily apparent to the skilled person. Itwill be appreciated that HCT values of calibrated blood samples may beobtained via other known methods to enable such calibration. Using thismethod the standard error of the estimated HCT values, when comparedagainst the known micro-haematocrit method, is found to be less than1.5% when measuring HCT in the range of 20 to 60%.

In a modified version of the embodiment of FIG. 1, one drive electrode(4) of the electrodes in the sampling zone (2) may possess a deposit ofan enzyme (e.g. glucose oxidase, glucose dehydrogenase) to react withfree glucose in the plasma component of the blood sample tosubstantially oxidise it. In that case the control processor is arrangedto control the electrical current generator to apply an alternatingelectrical current at a first frequency (selected from within the secondrange of values) with a DC voltage offset (e.g. about 0.25 volts)applied across the drive electrodes (3, 4) and concurrently to controlthe voltage detector (21) to measure a first voltage signal received viathe first sensing electrode, and to measure a second voltage signalreceived via the second sensing electrode. Subsequently, the controlprocessor further controls the electrical current generator to apply analternating electrical current at the same first frequency (selectedfrom the second frequency range) without a DC voltage offset appliedacross the drive electrodes and concurrently to control the voltagedetector (21) to measure a third and a fourth voltage signal valuereceived from the first and second sensing electrodes respectively.

The control processor is arranged to calculate a first electricalreactance value (X₁, Ohms) using an amplitude of the voltage differencebetween the first and second voltages measured at the first frequencyand the amplitude of the associated applied alternating current, and tocalculate a second electrical reactance value (X₂, Ohms) using anamplitude of the voltage difference between the third and fourthvoltages measured at the second frequency, and the amplitude of theassociated applied alternating current.

The control processor then calculates a value (HbA1c) representing theconcentration of glycated haemoglobin within the blood sample accordingto the following equation.

${HbA1c} = {100 \times \left( {1 - \frac{X_{1}}{HCT \times X_{2}}} \right)}$

Where HCT is a predetermined haematocrit value for the blood sampleobtained independently, e.g. according to the invention, or otherwise.

This method has been found to provide an HbA1c measurement result withan accuracy of ±10% or better within 20 seconds. The concentration ofHbA1c depends on both the concentration of glucose in the blood and thelifespan of the erythrocyte (the haemoglobin cell). Because erythrocytesare in circulation for approximately 120 days HbA1c represents theintegrated glucose concentration over the preceeding 8 to 10 weeks,which is therefore free of the large fluctuations that occur daily inblood glucose concentrations in blood plasma.

Before measurement of a sample of blood is performed, the temperature ofthe sampling plate is established. This may be done by any suitablemeans such as would be available and apparent to the skilled person.Preferably, the temperature of the sampling plate may be determinedmeans of a thermocouple mounted in the strip port connector. To furtherimprove accuracy the temperature of the sample should preferably bemaintained at 37° C.±1.5° C. This may be achieved, for example, byenvironmental temperature control of the area in which sampling platesare stored or used, or by means of a heater (e.g. trace heating, Ohmicheating wire/strip etc, not shown) formed in the strip electricallyconnectable to a power source within the sensing unit (15, FIG. 1) tocontrollably heat the sampling plate. A thermocouple (not shown) mayalso be formed within/upon the sampling plate, also being arranged to bepowered by the sampling unit when the sampling plate is connectedthereto in use. This may be used to regulate the heater (if present)and/or simply to allow the sampling unit to determine the temperature ofthe sampling plate.

It will be noted that both the reactive (X₃) and resistive (R₂)components of the impedance value (Z₂) are employed in these equationswhen employing the higher frequency AC signals, whereas only a resistivecomponent (R₁) is used at lower signal frequencies. This stems from aconsideration of the electrical current paths which may be considered toflow across the linear arrays of blood in the sample received within thesampling and partitioning gaps of the sample zone as follows.

FIG. 5 illustrates an equivalent circuit representing what is postulatedto be the conductive pathways for electrical current driven through ablood sample between drive electrodes of the sampling plate. This ispostulated, but not asserted, as it is useful to understanding.

A first current pathway (100) passes current across the line of bloodcells (101) within the sensing gap through the blood plasma (102), orother added liquid, between blood cells. This conductive path can beconsidered as purely resistive in nature. A second conductive path (103)passes through a blood cell (104). The path through the contents of theblood cell (e.g. any glucose) may be considered as resistive, whereasthe path through the walls of the blood cell may be consideredcapacitive.

These two current pathways act in parallel and present an electricalimpedance (Z) which may be approximated as follows.

$Z = {{R + {jX}} = {R_{a}\left\{ \frac{1 + {\omega^{2}C_{b}^{2}{R_{b}\left( {R_{b} + R_{a}} \right)}} - {j\;\omega\; C_{b}R_{a}}}{1 + {\omega^{2}{C_{b}^{2}\left( {R_{b} + R_{a}} \right)}^{2}}} \right\}}}$

Where R_(a) is the resistance of the plasma, R_(b) is the resistance ofthe contents of the blood cell including any glucose, and C_(b) is thecapacitance of the blood cell, where j=√{square root over (−1)}.

At sufficiently low frequencies, the reactive impedance of the bloodcell, arising from the capacitance of the blood cell, is very high andprevents current flow through the cell. Substantially only the firstcurrent path (through plasma) is available. At sufficiently highfrequencies, the reactive impedance of the blood cell falls and thesecond current pathway becomes increasingly significant. The secondpathway brings the influence of the resistance of the content of a bloodcell to bear on the value of the impedance Z as well as the remaininginfluence of the plasma due to the remaining first current pathway. Inthis way, employing the components of impedances of a blood sample atboth low and high frequencies enables the influence of the contents ofthe blood cell to be probed.

It has been found that the presence of glucose within a blood cell has ameasurable effect upon the phase of the electrical current signalpassing through the sample. It is postulated that this may be becausethe presence of glucose within the blood cell increases the number ofelectrons available to react to the oscillating drive signal therebyincreasing the flow of current through the cell. This influences thephase of the current passing through the sample. The presence of glucosein the cell can be considered as influencing the resistance R_(b) of thecell contents, according to the equivalent circuit model. The phase of avoltage sensed in the blood sample under such circumstances could berepresented as:

${Phase} = {{\arctan\left( \frac{X}{R} \right)} = {\arctan\left( \frac{\omega\; C_{b}R_{a}}{1 + {\omega^{2}C_{b}^{2}{R_{b}\left( {R_{b} + R_{a}} \right)}}} \right)}}$

The phase angle can be seen to be influenced by R_(b), the resistance ofthe cell contents. Thus, it is postulated that this may be part of theorigin of the relationship between the phase of an electrical drivesignal within the sample, and the amount of glucose within the bloodcells (i.e. relating to HbA1c).

FIG. 2 illustrates a close-up view of the electrode terminals (3, 4, 5and 6) of FIGS. 1, 3 and 4. The figure indicates a suitable sensing gapwidth and partitioning gap widths either side of the sensing gap. Asensing gap of 100 microns uniform in width is suitable. Partitioninggap widths of 150 microns is also suitable. Each sensing electrodeterminal within the drive gap, between the two drive electrode terminals(3, 4) is a straight-edged flat strip of Gold having a substantiallyuniform width of 200 microns. This results in a drive gap width of 800microns. Similarly, each drive electrode terminal is a flat segment ofGold.

FIG. 3 illustrates an embodiment of a sensing plate of FIG. 1 in theform of a disposable strip (1). The electrode and conductor structure ofthe disposable strip is as described above with reference to FIG. 1. Inaddition, the end of the strip containing the drive and sensingelectrode terminals (3, 4, 5 and 6) comprises sample zone (2) forreceiving the blood sample, surrounded by an air-porous body (27) whichis in fluid communication with the sample zone wherein the air porousbody is arranged to receive air displaced from the sample zone as theliquid blood sample is received into the sample zone.

“In fluid communication with” may mean interfacing, where “interfacing”means sharing a common boundary. Preferably “in fluid communicationwith” refers to where the air porous body is adjacent to the samplezone. The air porous body may define a floor of the sample zone and/orwall(s) of the sample zone. The air porous body may surround the samplezone. Preferably the air porous body defines the sample zone, or definesan outer boundary of the sample zone. Preferably the air porous bodydefines the perimeter of the sample zone or at least part of theperimeter of the sample zone. Preferably the air porous body is externalto the sample zone itself. Preferably the sample zone is free of airporous body.

Preferably the air porous body is arranged to receive displaced air asthe liquid sample approaches the air porous body. Preferably the airporous body is arranged to receive air displaced in the same directionas the liquid sample travels (or spreads) into the sample zone.Preferably the air porous body is arranged to receive a side-waysdisplacement of air as the liquid sample approaches the air porous bodyin a side-ways manner. Preferably the sample zone is arranged to preventback flow of the liquid sample.

An advantage of this arrangement is that the air porous body helps theliquid sample to flow into the sample zone with minimal air resistance,by providing a means by which air can be directly displaced—preferablyin the same direction as the liquid sample enters the sample zone. Thispermits the liquid sample to enter the sample zone at a faster rate. Incontrast, where such an air porous body is absent, air resistanceretards the flow of the liquid sample into the sample zone.

Another advantage of the arrangement is that the air porous body helpsthe liquid sample to spread uniformly throughout the sample zone, thusgiving greater sampling consistency and consequently more accuratemeasurements. In contrast, where the air porous body is absent, airresistance affects the fluid dynamics of the liquid sample bydiscouraging spreading (air resistance from all sides) and insteadencouraging the liquid sample to remain collectively associated as abulk (aided by surface tension). As such the liquid sample tends to flowas a bulk in a single direction since in this way the bulk overcomes airresistance in that particular direction. Another advantage is thatformation of air-pockets is alleviated, which again allows for betterspreading and more accurate measurements. The liquid sample ispreferably hydrophilic, more preferably aqueous-based, and mostpreferably blood. In this case, blood glucose levels of a diabeticpatient may be measured. The air porous body is preferably substantiallyimpermeable to the liquid sample. The air porous body is preferablysubstantially impermeable to water. The air porous body is preferablysubstantially impermeable to an aqueous liquid sample, and mostpreferably substantially impermeable to blood.

The air porous body is preferably located substantially around theperimeter of the sample zone. Preferably a floor of the sample zone isfree of air porous body. Preferably the sample zone is free of a roof.Where the sample zone comprises a roof, the roof is preferably free ofair porous body. The air porous body preferably comprises hydrophobicmaterial. Preferably the air porous body comprises at least 50 wt %,more preferably at least 70 wt %, and most preferably at least 90 wt %hydrophobic material. The air porous body preferably has an average poresize between 10 and 300 microns, preferably between 50 and 200 microns,and most preferably between 100 and 150 microns. The air porous bodypreferably comprises an air porous mesh, which again is preferablyhydrophobic overall. Such an air porous mesh preferably comprisespolyether ether ketone (PEEK), polypropylene (PP), polyester (PET),polyvinylidene fluoride (PVDF), ethylene chlorotrifluoroethylene(ECTFE), ethylene co-tetrafluoroethylene (ETFE), nylon (polyamide), orfluorinated ethylene-propylene (FEP). The air porous mesh preferablycomprises polyester (PET). Most preferably the air porous mesh comprisesSefar 07-120 34.

Accordingly, where the sample zone (2) has a roof, the sample zone isaccessible via an entry port (25) into which a blood sample (26) may beplaced. By capillary action, the blood sample is drawn through the entryport and into the sampling zone, displacing air into the air-porous body(27) as it does so, to finally occupy the sample zone covering the driveand sensing electrode terminals there. A breathable structure createdbeneath a thin polymer film covering the sample zone, as a roof.Typically the porous layer is a mesh made up of strands of polymer thatare coated to create a hydrophobic boundary to the blood as it flows onto the sample zone. A geometric shape cut into the mesh defines thesample zone and entry port which allows the sample to fill the samplezone under capillary action created by the thin top film.

FIG. 4 illustrates a further embodiment of the invention in which asampling plate (30) comprises two sets (31, 32) of substantiallyidentical drive electrode and sensing electrode arrangements each beingsubstantially identical in structure as the electrode and conductorstructure described above with reference to FIGS. 1 to 3. In particular,a first electrode group (31) comprises a pair of aforesaid driveelectrode terminals (3 a, 4 a) located within a first sample zone (2 a)of the sampling plate. A pair of aforesaid sensing electrode terminals(5 a, 6 a) extend in parallel along the drive gap formed between the twodrive electrodes. A respective one of four conductive strips (7 a, 8 a,9 a, 10 a) electrically connects a drive terminal or sensing electrodeterminal to a respective one of four separate contact zones (11 a, 12 a,13 a, 14 a) arranged along the distal edge of the strip. A secondelectrode group (32) comprises a pair of aforesaid drive electrodeterminals (3 b, 4 b) located within a second sample zone (2 b) of thesampling plate. A pair of aforesaid sensing electrode terminals (5 b, 6b) extend in parallel along the drive gap formed between the two driveelectrodes. A respective one of four conductive strips (7 b, 8 b, 9 b,10 b) electrically connects a drive terminal or sensing electrodeterminal to a respective one of four separate contact zones (11 b, 12 b,13 b, 14) arranged along the distal edge of the strip. Accordingly,eight contact zones arrayed along the distal edge of the sampling stripfor insertion into a socket/port of an electrical sensing unit (42) toplace the each one of the eight contact zones simultaneously inelectrical connection with a respective one of eight electrical contactterminals of the sensing unit.

The first and second sample zones (2 a, 2 b) of the sampling plate areeach in communication with a common single sample entry port (35) via arespective one of two sample conduits (36, 37) which bifurcate from theentry port and communicate with a given sample zone. The two samplezones (2 a, 2 b) are each surrounded by an air-porous body (27), asdescribed above, which is in fluid communication with each of the samplezones wherein the air porous body is arranged to receive air displacedfrom the sample zone as the liquid blood sample is received into thesample zone. The air porous body defines the entry port (35) and thesample conduits (36, 37) as well as the circular periphery of each ofthe two sample zones.

A quantity of an enzyme (38), (e.g. glucose oxidaze (“GOX”) or glucosedehydrogenase (GDH)), is located upon one of the two drive electrodeterminals (4 b) in the second sample zone (2 b). The enzyme (e.g. GOX orGDH) is placed to allow it to make contact with, and react with, a bloodsample entered into the second sample zone. In doing so, the enzymereacts with the blood sample to consume any free glucose present withinthe plasma of the blood sample. As a result, one the reaction hascompleted, the blood sample located within the second sample zonecontains substantially no free glucose, and any glucose present shouldbe substantially only fixed glucose within the red blood cells of theblood sample which is inaccessible to the enzyme (e.g. GOX or GDH).

In the sensing unit (42), a control processor (41) is arranged tocontrol the current source (18) and the voltage detector unit (21) asdescribed above with reference to FIG. 1 when measuring haematocrit(HCT). The control processor (41) is arranged to control a secondcurrent source (39) and a second voltage detector unit (40) of thesensing unit, as described above with reference to FIG. 1 (“modifiedversion”) when used to measure HbA1c.

FIG. 6 schematically illustrates an ASIC (application specificintegrated circuit, 50) arranged for connection to a microcontroller(55) integrated circuit for use within the sensing unit (42, FIG. 4).The ASIC is responsive to the control signals from a control unit (55)to apply to the second group of electrodes (31) of the sensing strip analternating (AC) current having a first frequency of 1 MHz and toselectively apply a direct voltage (DC) of most preferably asubstantially constant value concurrently, accordingly to the controlsignals. This is for the purposes of measuring HbA1c in the blood samplebridging the electrodes of that second group.

The ASIC is responsive to the control signals from a control unit (55)selectively to apply to the first group of electrodes (32) of thesensing strip an alternating (AC) current having either a firstfrequency of 1 MHz or second frequency of 50 KHz according to thecontrol signals. This is for the purposes of measuring haematocrit inthe blood sample bridging the electrodes of that second group.

The ASIC is arranged to receive voltage signals from the first andsecond sensing electrode terminals (5 b, 6 b) of the second group ofelectrodes (31) of the sensing plate, and to receive voltage signalsfrom the first and second sensing electrode terminals (5 a, 6 a) of thefirst group of electrodes (32) of the sensing plate. The ASIC isresponsive to the voltage signals and the current signals to measurepeak/amplitude values for those voltages and currents for use by themicrocontroller in calculating electrical resistance and reactancevalues of blood samples bridging the electrodes of the first and secondelectrode groups (31, 32) to determine values of haematocrit and HbA1ctherein.

The control unit (55) performed the functions of the control processorunit (41) of FIG. 4.

In particular, the microcontroller (55) is arranged to provide a direct(DC) voltage level (most preferably a substantially constant (DC)voltage level) and to output a corresponding (DC) analogue output signalvia a digital-to-analogue converter output (56) connected to an inputport (57) of the ASIC (50). Furthermore, the microcontroller is alsoarranged to produce an alternating (AC) pulse-width modulatedsquare-wave analogue output signal via a pulse-width modulator (PWM, 58)as a first signal (59) having a frequency of 1 MHz and to input thesignal to a second input port (61) of the ASIC, and to produce a secondseparate modulated square-wave output signal (60) having a frequency of50 KHz and to input the signal to a third input port (62) of the ASIC.These two pulse-width modulated output signals are electrical currentsignals which are each maintained at a predetermined amplitude level(most preferably substantially constant) by the microcontroller. TheASIC includes a first pre-amplifier in the form of an operationalamplifier (63) comprising a feed-back loop (64) including a resistor.The pre-amplifier is arranged to receive the DC voltage signal from themicrocontroller at a first input port (67) of the pre-amplifier, and tooutput an amplified value to a first output port (65) of the ASIC. TheASIC also includes a 1 MHz sinewave filter unit (67) and a 50 KHzsinewave filter unit (68) each arranged for receiving and filtering arespective 1 MHz and a 50 KHz oscillating current signal input from themicrocontroller at the second and third input ports (61, 62) of theASIC. Each of the sinewave filter units is arranged to receive therespective square-wave (PWM) signal input to it, and to alter thesquare-wave shape of the signal into substantially a sine-wave shape andto output the result as an AC sine-wave signal. An analogue switch unit(69) is provided in the ASIC and has two input ports for receiving arespective one of the two AC sinewave signals output from the 1 MHz and50 KHz sinewave filter units. The analogue switch unit is arranged to becontrolled by the microcontroller to output selectively one of the 1 MHzsinewave (AC) signal and the 50 KHz sinewave signal to a signal inputport of a second pre-amplifier (71) in the ASIC for amplifying thatoutput signal. In this way the microcontroller is able to control, viathe analogue switch unit, which of the 1 MHz and the 50 KHz sinewave(AC) signals is ultimately output from the second pre-amplifier and fromthe ASIC.

The sampling strip (30) is shown as electrically connected to the ASICof the sensing unit such that a first drive electrode terminal (4 b) ofa second group of electrodes (31) for sensing HbA1c, is electricallyconnected to the first output port of the ASIC, and such that a firstdrive electrode terminal (4 a) of a first group of electrodes (32) forsensing haematocrit, is electrically connected to the second output portof the ASIC.

In addition, the analogue switch unit is arranged to be controlled bythe microcontroller to output the 1 MHz sinewave (AC) signal to a secondsignal input port (66) of the first pre-amplifier (63) in the ASIC foramplifying that output signal. The first pre-amplifier unit is arrangedto selectively amplify a 1 MHz sinewave (AC) signal either with orwithout the concurrent presence of the DC voltage level applied to thefirst input port (67) of that amplifier. The microcontroller controlswhen/if the DC voltage level is applied to the first pre-amplifier unit.

The analogue switch unit is further arranged to output, via a thirdoutput port (73), a signal which is representative of the time of apeak/amplitude of the AC sinewave signal received by the analogue switchunit from either of the first and second sinewave filter units. Thissignal is input to a phase-to-voltage unit (74) which, as shall beexplained in more detail below, is arranged to measure a temporal phasedifference between an AC current applied to the drive electrodes of thefirst and second groups of electrodes, and an AC voltage generatedbetween sensing electrode terminals of the respective groups ofelectrodes of the sensing plate, and to generate the result as a signalrepresentative of that temporal phase difference.

The ASIC includes a peak detector unit (77) in communication with thephase-to-voltage unit and comprising a first and second signal inputports (82, 83) in communication with first and second voltage inputports (75, 76), respectively, of the ASIC for receiving voltage signalsfrom the sensing electrode terminals (5 a, 6 a) of the first group ofelectrodes (32) of the sensing plate. A third input port (84) of thepeak detector unit is arranged in communication with a second driveelectrode (3 a) of the first group of drive electrodes (32) via a firstcurrent input port (78) of the ASIC and is arranged to receive thecurrent driven through the first group of electrodes between the firstdrive electrode (4 a) and the second drive electrode (3 a) of thatgroup.

The peak detector unit also comprises a fourth and fifth signal inputports (85, 86) in communication with third and fourth voltage inputports (79, 80), respectively, of the ASIC for receiving signals from thesensing electrode terminals (5 b, 6 b) of the first group of electrodes(31) of the sensing plate. A sixth input port (87) of the peak detectorunit is arranged in communication with a second drive electrode (3 b) ofthe first group of drive electrodes (31) via a second current input port(81) of the ASIC and is arranged to receive the current driven throughthe second group of electrodes between the first drive electrode (4 b)and the second drive electrode (3 b) of that group.

Thus, the ASIC includes a first (75) and second (76) voltage input portswhich connect electrically to a first (82) and second (83) input portsof a peak detector unit and are adapted for receiving a respective firstand second voltages from respective of the sensing electrodes of thefirst group of electrodes (32). The ASIC also includes a third andfourth voltage input ports (79, 80) which are similarly electricallyconnected to input ports (85, 86) of the peak detector unit. A firstcurrent input port (78) and a second current input port (81) are eachrespectively connected to first and second current input ports (84, 87)of the peak detector unit. Each of the first to sixth input ports of thepeak detector unit are arranged to receive respective voltage andcurrent signals which are amplified by a respective pair of amplifierunits (92) within the apparatus, arranged in series electricalconnection along the respective signal transmission line leading to theinput ports in question. These amplifier units serve to amplify therespective signals prior to receipt by the peak detector unit, and maybe formed in the ASIC if desired, or elsewhere in the sensing unit butoperably connected to the ASIC as shown in the Figures.

The peak detector unit comprises an output port electrically connectedto a sample-and-hold unit (90) which is arranged to receive from thepeak detector unit a signal representing a value of the peak in an ACvoltage signal received by the peak detector, and/or a valuerepresenting a peak current value received by the peak detector. Thesepeak values generally represent amplitude values of an associated ACcurrent and voltage signal inputs to the peak detector unit from theelectrodes of the sampling plate (bio-sensor module). Thesample-and-hold unit is operable to momentarily retain signal valuesreceived from the peak detector unit as and when required for subsequenttransmission to the microcontroller.

The phase-to-voltage unit (74) possesses an output port electricallyconnected to an output port of the ASIC for outputting a voltage signalrepresenting a measured phase difference (measured time shift) betweenthe peak voltage values detected by the peak detector, and the peakvalues of the AC current signal output by the analogue switching unit.

A stop-watch unit (89) is operably connected to and controlled by themicrocontroller (55) via the control register unit (91) and theinterface (SPI) with the microcontroller. The stop-watch unit isarranged to measure a time interval between detected signal peaksdetected by the peak detector unit for use in measuring a phase anglebetween applied current and measured voltage.

In this way, the phase-to-voltage unit and the peak detector unitprovide values representing an amplitude of an AC voltage signal and anAC current signal received by the peak detector unit, and a phasedifference incurred between those signals. With these measured values,the microcontroller is arranged to calculate values of resistance andreactance of samples within the sampling zones of the sampling plate(bio-sensor module).

The sample and hold unit (90) has two output ports each in communicationwith a respective first and second output port (94, 95) of the ASIC forseparately outputting values associated with the first and second groupsof electrodes (31, 32) respectively.

The voltage output port of the ASIC is arranged to be connectable (andis shown as connected to) a biosensor module in the form of a samplingstrip such as is shown in FIG. 4. The first current output port (65) ofthe ASIC is connected to a drive electrode (4 b) in the sampling zone ofthe sampling strip dedicated to measuring HbA1c and the second currentinput port (81) is arranged to be electrically connected to (and isshown as connected) to the other drive electrode (3 b) in that samplingzone. Similarly, the second current output port (72) of the ASIC iselectrically connectable to (and shown as connected) a first driveelectrode (4 a) in the other sampling zone of the sampling stripdedicated to measuring haematocrit, whereas the other drive electrode (3a) in that sampling zone is electrically connectable to (as is shown asconnected) the first current input port (78) of the ASIC.

The two sensing electrodes (5 a, 6 a) of the sampling strip within thesampling zone for haematocrit are each separately connected to the firstand second voltage input ports (75, 76), respectively, of the ASIC via arespective pair of amplifier units (92). Similarly, the two sensingelectrodes (5 b, 6 b) in the sampling zone of the sampling stripdedicated to measurement of HbA1c are each separately connected to arespective one of the third and fourth voltage input ports (79, 80) ofthe ASIC. In this way, electrical currents may be driven across thedrive electrodes in the two sampling zones via the ASIC, and resultingvoltages, and voltage/current phase differences arising from liquidsamples within those sensing zones, may be determined.

In particular, when an AC current signal is driven across a pair ofdrive electrode terminals of the first or second group of electrodeterminals, a blood sample bridging the drive gap between those electrodeterminals responds in such a way as to present an electrical impedance(Z, ohms). This manifests itself in that an AC electrical potentialdifference is generated across the sample, as measured by/between thetwo sensing electrode terminals in the given group of terminals, whichis not in temporal phase with the AC current signal. The samplepossesses not only a resistance (R, ohms) which is dissipative (real),but also a reactance (X, ohms) which is non-dissipative (imaginary).

Thus, if the driving AC current of amplitude |I| and angular frequency ωis represented by:I=|I|exp{j(ωt+θ ₁)}where j=, √{square root over (−1)}, and θ₁ is the temporal phase of thesine wave AC current signal, then the resulting voltage across the twosensing electrodes of the given group of electrodes is represented by:V=|V|exp{j(ωt+θ _(V))}where θ_(V) is the temporal phase of the sine wave AC voltage signal ofamplitude |V| and angular frequency ω.

The impedance of the sample is given by:Z=|Z|exp{jϕ}where |Z| is the magnitude of the impedance and ϕ is its phase angle.

The resistance of the blood sample (R) is given by:R=|Z|cos(ϕ)

And the reactance of the blood sample (X) is given byX=|Z|sin(ϕ)where |Z| is measurable using the known amplitudes |I| and |V| of theapplied current and resulting voltage signals respectively. Similarly,the phase angle of the impedance can be determined by applying Ohm'slaw:V=|V|exp{j(ωt+θ _(V))}=I×z=|I|×|Z|exp{j(ωt+θ ₁+ϕ)}thusϕ=θ_(V)−θ_(I) =ω×Δtwhere Δt is the time lag between a peak in the applied AC sinewavecurrent signal and a measured peak in the resulting AC voltage acrossthe blood sample.

Thus, the resistance (R) and reactance (X) values of the blood samplemay be determined from the AC current peak/amplitude value (|I|) asapplied at a frequency ω between the two drive electrode terminals in asampling zone, the resulting AC voltage peak/amplitude (|V|) as measuredbetween the sensing electrodes in that sampling zone, and the time lag(Δt) between those two peaks in succession, as follows:

$\begin{matrix}{R = {{\frac{V}{I}}{\cos\left( {\omega \times \Delta\; t} \right)}}} & {{Equation}\mspace{14mu}(1)} \\{X = {{\frac{V}{I}}{\sin\left( {\omega \times \Delta\; t} \right)}}} & {{Equation}\mspace{14mu}(2)}\end{matrix}$

These values are either known by, or measured by, the sampling unitusing the ASIC as described above, and the microcontroller (55) isarranged to calculate these resistance and reactance values.

A control register unit (91) is provided on the ASIC and is arranged tocontrol, under overall control of the microprocessor via an interfaceunit (SPI), the timings and orchestration of control signals foroperation of the amplifiers (/en signals to “enable” amplifiers),filters, switches and other components on the ASIC. The control registermay be of a type such as would be readily apparent to the skilledperson.

A thermo-couple (88) is formed in the ASIC and is electrically connectedbetween a signal port of the control register unit (91) and an earthterminal and is arranged in the sampling device to make physical contactwith a part of the sampling plate when the sampling plate is operativelyconnected to the sampling device in use. The control register unit isarranged to receive signals from the thermo-couple representative of thetemperature of the sampling plate (30) and to convey those signals tothe microcontroller to permit the temperature of the sampling plate tobe determined by the microcontroller.

In use, the sampling unit is operable as follows. Once a user hasapplied a blood sample to the first and second sampling zones (FIG. 4; 2a, 2 b) of the sampling plate (30), the blood sample is split betweenthe first sampling zone (2 a) dedicated to measuring haematocrit, andthe second sampling zone (2 b) dedicated to measuring HbA1c. The GOx/GDHspot (38) provided in the second sampling zone reacts with the freeglucose present in the blood plasma of the blood sample within that zoneto substantially oxidise it. A suitable period of time is allowed forthis process (several seconds, e.g. between 0.5 and 15 seconds). Thishas been found to be as little as 0.5 seconds. It may be slowed by usinga mediator which would allow the signal transient to be recorded over atime frame sufficient to determine the level of free glucose in thesample. For the determination of HbA1c, it is only required that thefree glucose is oxidised, therefore it is important at least to see thata transient has occurred and decayed before the HbA1c measurementsequence is started. Subsequently, the blood-bearing sampling strip iselectrically connected to the sampling unit (if not already soconnected) as shown in FIG. 4 and FIG. 6.

The microcontroller shown in FIG. 6 is arranged to supply to the firstpre-amplifier (63) of the ASIC (50) an alternating (AC) electricalcurrent having a 1 MHz frequency, via the 1 MHz sinewave filter unit(67), to drive that current through the blood sample bridging the twodrive electrodes within the second sampling zone, thereby to generate afirst alternating potential difference across the spacing between thedrive electrode terminals measurable via the two sensing electrodeterminals (5 b, 6 b) within that sampling zone. This state is maintainedfor a period of time of several milliseconds in duration (e.g. betweenabout 20 ms and about 200 ms or less). Subsequently, the microcontrolleris arranged to apply a substantially constant direct (DC) voltage to thefirst pre-amplifier unit (63) of the ASIC to output to the first outputport of the ASIC (65) a substantially constant DC offset of about 0.25volts in combination with the 1 MHz AC current signal concurrently beingapplied to the second sampling zone. The microcontroller is arranged tothen employ the peak detector unit and the phase-to-voltage unit of theASIC to measure the amplitude of the voltage across the two sensingelectrode terminals (5 b, 6 b) in the first sampling zone, and the timelag between the successive occurrences of a peak in the applied ACcurrent and the resulting AC voltage, and to measure a first value ofthe electrical reactance (X₁) of the blood sample according to Equation(2) above.

The microcontroller is arranged to subsequently not apply the DC voltageoffset and to continue to apply the 1 MHz AC current signal to the firstand second drive electrode terminals of the second sampling zone. Aftera suitable time period following removal of the DC offset voltage (e.g.between about 20 ms and about 200 ms or less), the microcontroller isarranged to then employ the peak detector unit and the phase-to-voltageunit of the ASIC to measure the amplitude of the voltage across the twosensing electrode terminals (5 b, 6 b) in the first sampling zone, andthe time lag between the successive occurrences of a peak in the appliedAC current and the resulting AC voltage, and to measure a second valueof the electrical reactance (X₂) of the blood sample according toEquation (2) above.

Either before, during or after performing the above process on the bloodsample within the second sampling zone, the microcontroller is arrangedto determine the haematocrit (HCT) within the blood sample as follows.

The microcontroller is arranged to supply a 50 KHz AC current to theanalogue switch unit (69) of the ASIC via the 50 KHz sinewave filterunit (68) and to control the switch unit to connect the 50 KHz ACcurrent to the second output port (72) of the ASIC via the secondpre-amplifier unit (71). This causes the AC signal to be applied to thedrive electrode terminals (3 a, 4 a) of the first sample zone, analternating electrical current having a first signal frequency of 50 KHzto generate a first alternating potential difference of 50 KHz acrossthe spacing between the electrode terminals, as measured by the twosensing electrodes (5 b, 6 b) in the first sample zone.

The microcontroller is arranged to then employ the peak detector unitand the phase-to-voltage unit of the ASIC to measure the amplitude ofthe voltage across the two sensing electrode terminals (5 a, 6 a) in thefirst sample zone, and the time lag between the successive occurrencesof a peak in the applied AC current and the resulting AC voltage, and tomeasure a first value of the electrical resistance (R₁) of the bloodsample according to Equation (1) above.

The microcontroller is arranged to then supply a 1 MHz AC current to theanalogue switch unit (69) of the ASIC via the 1 MHz sinewave filter unit(67) and to control the switch unit to connect the 1 MHz AC current tothe second output port (72) of the ASIC via the second pre-amplifierunit (71). This causes the 1 MHz AC signal to be applied to the driveelectrode terminals (3 a, 4 a) of the first sample zone, an alternatingelectrical current having a second signal frequency of 1 MHz to generatea second alternating potential difference of 1 MHz across the spacingbetween the electrode terminals, as measured by the two sensingelectrodes (5 a, 6 a) in the first sample zone.

The microcontroller is arranged to then employ the peak detector unitand the phase-to-voltage unit of the ASIC to measure the amplitude ofthe voltage across the two sensing electrode terminals (5 a, 6 a) in thefirst sample zone, and the time lag between the successive occurrencesof a peak in the applied AC current and the resulting AC voltage, and tomeasure a second value of the electrical resistance (R₂) of the bloodsample according to Equation (1) above, and to measure a third value ofthe electrical reactance (X₃) of the blood sample in the first samplezone according to Equation (2) above.

The microcontroller is arranged to subsequently calculate a value forthe relative volume of red blood cells (haematocrit, HCT) in the liquidsample according to the first electrical resistance value, the secondelectrical resistance value and the third electrical reactance valueaccording to the following formula, and store the result and/or tooutput the result to the user:

$\begin{matrix}{{HCT} = \left\lbrack {{A\;{\ln\left( \frac{R_{1}}{R_{2}} \right)}} + {B\;{\ln\left( {X_{3} + X_{0}} \right)}} - C} \right\rbrack} & {{Equation}\mspace{14mu}(3)}\end{matrix}$

Where A, B and C are constants associated with the sampling plate inquestion. For example, the values of A, B and C may each typically bewithin the range from about 0.05 to about 0.5, or preferably betweenabout 0.1 and 0.25, or more preferably between about 0.1 and about 0.2.For example, when using electrodes formed from gold having a sheetresistance of 5 ohms per square, and the geometry illustrated in FIG. 4,the values in question may be:

A=0.142;

B=0.155;

C=0.157.

Actual values, suited to a given sampling zone geometry and electrodestructure and material, may be determined by routine calibrationemploying commercially available blood samples of known HCT, as will beapparent to the skilled person. The value of X₀ may simply be zero, ormay be adjusted if necessary to improve the predictive accuracy of theequation. It has been found that the term A is correlated with theconductivity of the electrode terminals of the sensing strip. The term Bhas been found to correlate with the resistivity of the interface (e.g.the wetting) between the blood sample and the electrode terminals in thesample zone. This is influenced by the electrode material (e.g. Gold),and the quality of the structure (e.g. roughness) of the electrodesurfaces. The term C has been found to correlate with the variability ofthe average blood cell size (e.g. determined for “unfixed” orun-glycated blood cells) within the sample. This can be stronglyinfluenced by ethnicity, blood type and interferences such as thosewhich will be readily apparent to the skilled person. Values of Cassociated with different ethnicities (or blood type or knowninterferences) may be stored in a look-up table within (or accessibleby) the control processor for suitable selection during calibration.

Table 1 shows examples of values of haematocrit for a blood sample oftype A calculated according to the above method and equations. Threegroups of ten measurements were made using the strip design illustratedin FIGS. 1, 3, 4 and 6 for measuring HCT. In each of the three groups asample of blood was used having a known HCT value, namely 52%, 42% and31%. Measurements of resistances R₁, and R₂ and reactance X₃ were madeat 50 KHz and 1 MHz AC signal frequency respectively, and input intoEquation (3) to generate a measurement value of HCT in each of themeasurements. The ten measurements for each one of the three differentcommon (known) HCT value shows a consistently accurate HCT measurement.

The microcontroller is arranged to then generate a value representingthe concentration of glycated haemoglobin (HbA1c) in the blood withinthe sample according to the first electrical reactance value, the secondelectrical reactance value and a value of the relative volume of redblood cells in the liquid sample (haematocrit, HCT) according to thefollowing formula, and store the result and/or to output the result tothe user:

$\begin{matrix}{{{HbA}\; 1c} = {100 \times \left( {1 - \frac{X_{1}}{{HCT} \times X_{2}}} \right)}} & {{Equation}\mspace{14mu}(4)}\end{matrix}$

The quantity X₁ represents the reactance of a blood sample due toglycated red blood cells in the blood within the second sampling zonefrom which free glucose has been substantially oxidized by GOx/GDH,whereas X₂ represents the reactance of the whole blood sample in whichboth plasma and red blood cells contain glucose. The proportion of thatreactance due to red blood cells is determined by the (HCT)×(X₂)according to the haematocrit of the sample in the first sampling zone.

FIG. 7 shows an alternative form of sampling strip and sampling unit toread the sampling strip for the purposes of measuring haematocrit alone.

This alternative sampling strip (30A) comprises a first sampling zonecontaining a first group of electrodes identical to the electrode groupillustrated in FIGS. 1 to 3, and in the first sampling zone (2 a) of thesampling strip of FIG. 4 and FIG. 6. The first group of electrodescomprises a pair of drive electrode terminals (3 a, 4 a) defining adrive gap within which are arranged two sensing electrode terminals (5a, 6 a) for sensing a voltage drop across a blood sample when bridgingthe drive gap resulting from the an AC current driven between the twodrive electrode terminals. The first group of electrodes, and thecircuit elements of the ASIC (50A) with which they are arranged toelectrically connect (shown as connected in FIG. 7) are the same asthose elements of the ASIC (50) illustrated above in FIG. 6. Thosecircuit elements are arranged to operate, and be controlled by themicrocontroller (55A) substantially as described above with reference tothe first sample zone of the sample strip of FIG. 6 for determining ameasure of haematocrit using equation (3) above.

However, the second sampling zone of this alternative sampling stripcomprises a single pair of drive electrodes terminals (100, 101)defining between them a curved drive gap (102). The drive electrode pairis arranged to connect electrically to the ASIC to apply a DC electricalvoltage (preferably substantially constant in value) across the curveddrive gap when/if bridged by a blood sample within the second sensingzone. A deposit of a reagent, such as an enzyme or the like, to reactwith glucose in the blood sample (e.g. glucose oxidase (GOX) or glucosedehydrogenase (GDH)) is provided on the surface of the anode (e.g.electrode terminal 101) of the two drive electrodes. Like items areassigned like reference symbols as between FIGS. 6 and 7.

The ASIC (50A) in this alternative design differs from the ASIC of FIG.6 in that the analogue switch unit (69A) does not provide a 1 MHz ACcurrent input (66, FIG. 6) to the first pre-amplifier unit (63). Thefirst pre-amplifier unit (63A) does not possess a feed-back loop (64,FIG. 6) and simply receives from the microcontroller a DC voltage signal(most preferably substantially constant), which it amplifies and outputsthe result to a first voltage output port (65A) of the ASIC forelectrical connection to a drive electrode (100) of the second samplezone.

The DC voltage signal is output by the microcontroller (55A) via adigital-to-analogue converter (56) and input to an amplifier (63A)formed within the ASIC the amplified output of which is input to avoltage output port (65A) of the ASIC and to a first input port (104) ofan operational amplifier unit (103) formed in the ASIC. The operationalamplifier has a second input port (105) which serves as voltage inputterminal of the sensing unit respectively connected to an anode terminal(101) of the second pair of drive electrodes of the sample plate whenthe latter is connected to the former in use as shown in FIG. 7.

The operational amplifier has a respective output port (107) which isconnected to its second input port via a feed-back loop comprising aresistor (106). As a result, the DC drive voltage (Vdrive) applied tothe first input port of the operational amplifier by the first amplifierunit (63A) is expressed as a correspondingly substantially constantvoltage level at the second input port (105) of the operationalamplifier. This produces a controllably constant potential differenceacross the drive electrodes of the electrode pair (100, 101) in thesecond sample zone relative to a reference voltage (Vret). Consequentialconduction through a blood sample in the second sample zone of thesampling strip enters the operational amplifier electrically connectedto that blood sample. The result is that a measurable current isreceived by the operational amplifier at its second input port (105).This current is output on the output port (107) of the operationalamplifier via a voltage amplifier (109) to a 12-bit analogue to digitalconverter (97B) of the microcontroller for use thereby in determining avalue for the blood glucose level within the sample in the second samplezone, as described below. The measured current is also input from theoperational amplifier to an input port of the peak detector unit (77)which is arranged to detect the occurrence of a peak in the detectedcurrent from the second sample zone, and to communicate the time of thatoccurrence to the microcontroller (55A) via a 12-bit analogue to digitalconverter (97B) of the microcontroller for use thereby in determining avalue for the blood glucose level within the sample in the second samplezone, as described below.

It has been found that a direct (DC) voltage held between the two driveelectrodes of the second sample zone, and thereby applied across theblood sample located there during the enzyme (GOX/GDH) reaction period,will cause a time-varying current to pass through the blood sample.Variation is believed to result, in part, from the changing (falling)quantity of free glucose within the plasma of the sample resulting in achanging electrical resistance of the plasma component of the blood.Most preferably, the DC voltage is substantially constant forsimplicity, however non-constant DC voltages (e.g. smoothly falling orrising in a controlled way) could be employed if desired, though this islikely to complicate design and operation of the apparatus and so asubstantially constant DC voltage is preferred.

Starting with an initial rise (near instantaneous) in current to a peakvalue at a time “t_(peak)”, the observed quantity of current fallsmonotonically as glucose is increasingly oxidised in the blood plasma.Sample temperature affects the rate of decay of the current—lowertemperatures result in faster decay. It has been found that the rate offall of the observed current, following the peak current value, ischaracteristic of the amount of glucose originally present in the plasmaof the blood sample before the oxidisation process began. The observedcurrent decay is highly reproducible when the process is repeated. Thus,by performing this process initially with a sufficient plurality a bloodsamples each having an incrementally different, known quantity ofglucose in its plasma component, one may build-up a plurality ofreference curves of the type described above (or data setsrepresentative of them) from which a future blood plasma glucosemeasurement may be made by reference. That is to say, with the pluralityof reference curves (or representative data) one may perform acontemporaneous blood sampling operation as described above so as togenerate a measurable current varying generally according to the currentdecay curve described above. By measuring a particular current value ata selected time during that current decay (i.e. a point along thecontemporaneous current decay curve) one may subsequently identify ablood plasma glucose level associated with that current value as derivedfrom a reference curve. The contemporaneously measured blood plasmaglucose level may then be concluded to have the same glucose level. ALook-Up Table (LUT) or other storage may be used for this purpose. Theprocess may include measuring a contemporaneous value “I_(m)” of thedecaying current at a specified time “t_(m)” following the time“t_(peak)” at which the detected peak of the measured current occurs—thespecified time having also been used when generating the referencecurves. This current value then identifies the glucose value stored inthe LUT associated with the reference curve which had the same currentvalue at the same specified time in its current decay phase. The storedvalue from the LUT which matches a contemporaneous value will identifythe associated blood plasma glucose level so measured. The specifiedtime (t_(m)) may be between about 1 sec. and about 15 sec. Differentreference curves or LUTs may be used according to the measuredtemperature of the sensing strip, as determined by the thermocoupledescribed herein for example.

In this way, the microcontroller is arranged to apply a DC voltage tothe pair of drive electrodes (100, 101) in the second sample zone, andto measure the resulting current.

The sampling unit contains such a LUT as described above, and isarranged to compare respective contemporaneously measured current(decaying) values separately from the second sample zone, with storedreference current values, to identify the closest match (or interpolatebetween the closest two matches) and to retrieve an associated bloodplasma glucose value “BG_(raw)” from the LUT associated with that match.There may comprise as plurality of LUTs which may be respectivelyassociated with reference curves generated for a common specifiedtemperature of blood sample. The calculating unit may be arranged toselect the appropriate LUT based on the measured temperature of thesampling strip at the time of the measurement at hand. A thermo-couple(not shown) may be provided in the sampling unit to physically contactthe sampling plate (30A) in use and provide signals to which themicrocontroller is responsive to determine a temperature of the samplingplate and, from that determination, select the most appropriate LUT foruse as described above. Alternatively, through testing varioustemperatures, a temperature correction factor (T_(c)) may be determinedand used to compensate the measurement for temperature effects. One LUTfor Im and tm may then be used and the value retrieved from the LUTusing I_(m) and t_(m) may then be adjusted using the temperaturecorrection factor (T_(c)) as appropriate.

The microcontroller is arranged to produce an adjusted value“BG_(corrected)” for the blood plasma glucose level so retrievedaccording to:BG _(corrected)=ƒ(BG _(row) ,HCT)  Equation (5)where ƒ(BG_(row),HCT) is a predetermined corrective function of themeasured haematocrit value HCT for the sample in the first sample zone,and of BG_(row) which is an uncorrected blood plasma glucose valuemeasured for the blood sample in the second sample zone. The form of thefunction ƒ(BG_(row),HCT) of the predetermined corrective function may beselected by the user.

One example is of the form:ƒ(BG _(row) ,HCT)=BG _(row)−[m×(HCT)+c]where m is a positive or negative constant and c is a positive ornegative constant. These values may be evaluated by calibration againstcommercially available calibration blood samples containing known HCTand glucose levels. This functional form exploits the finding thaterrors in uncorrected glucose measurements are typically linear to afirst approximation, as a function of HCT, and that so too is thecorrective function. Of course, other more accurate correctivefunctional forms may be used such as would be apparent to the skilledperson in this field.

The ASIC controller unit (91) controls the timing and coordination ofthe components formed upon the ASIC under the master control of themicrocontroller via an interface (SPI) of the microcontroller with whichthe ASIC control unit is in communication. In this way, the requiredcurrent and voltage values may be applied to, and received from, thefirst sample zone containing the first group of electrodes (3 a, 4 a, 5a, 6 a) of the sampling plate (30A) via the ASIC to enable themicrocontroller to perform the measurements of HCT and blood glucose asdescribed above using equation (3).

This value of HCT is employed by the microcontroller to calculate anadjusted value of blood glucose in the sample of blood using the bloodglucose value determined from the second sampling zone containing thesecond group of electrodes (100, 101).

Examples of the HCT value determined by the microcontroller using theASIC and the first group of electrodes (3 a, 4 a, 5 a, 6 a) in the firstsampling zone, as described above with relation to FIGS. 1, 3 and 6, andemployed in Equation (3), are given in Table 1.

Aspects of the invention are defined by the following numberedparagraphs:

Paragraph 1: A sampling apparatus for use in performing electricalmeasurements on a liquid sample containing blood, the apparatuscomprising: two (or more) current output terminals for outputting analternating current signal applied therebetween; an alternatingelectrical current unit in electrical communication with the two (ormore) current output terminals for applying thereto an alternatingelectrical current of a given amplitude and frequency when a said liquidsample is in electrical connection between the two (or more) currentoutput terminals; a voltage unit in electrical communication with thetwo (or more) current output terminals for applying therebetween adirect (DC) electrical potential difference of a given magnitude; afirst voltage input terminal for receiving a first electrical signalexternally input thereto and a separate second voltage input terminalfor receiving a second electrical signal externally input thereto whensaid liquid sample is in electrical connection between the first andsecond voltage input terminals; voltage detector(s) for measuring afirst voltage and a second voltage using said first and secondelectrical signals, respectively; a control unit arranged to control theelectrical current unit to apply a said alternating electrical currentof said given frequency and concurrently to control the voltage unit andthe voltage detector(s) to measure said first and second voltages bothwhen said direct electrical potential difference is applied and whensaid direct electrical potential difference is not applied; acalculating unit arranged to calculate a first electrical reactancevalue using the first and second voltages measured when said directelectrical potential difference is applied, and to calculate a secondelectrical reactance value measured when said direct electricalpotential difference is not applied; wherein the calculating unit isarranged to generate a value representing the concentration of glycatedhaemoglobin (HbA1c) in the liquid sample according to the firstelectrical reactance value, the second electrical reactance value and avalue representing the relative volume of red blood cells in the liquidsample (haematocrit).

Paragraph 2: A sampling apparatus according to Paragraph 1 in which thegiven frequency has a value in the range 500 KHz to 1.5 MHz.

Paragraph 3: A sampling apparatus for use in performing electricalmeasurements on a liquid sample containing blood, the apparatuscomprising: two (or more) current output terminals for outputting analternating current signal applied therebetween; an alternatingelectrical current unit in electrical communication with the two (ormore) current output terminals for applying therebetween an alternatingelectrical current of a given amplitude and frequency, when a saidliquid sample is in electrical connection between the two (or more)current output terminals; a first voltage input terminal for receiving afirst electrical signal externally input thereto and a separate secondvoltage input terminal for receiving a second electrical signalexternally input thereto, when said liquid sample is in electricalconnection between the first and second voltage input terminals; voltagedetector(s) for measuring a first voltage and a second voltage usingsaid first and second electrical signals, respectively; a control unitarranged to control the electrical current unit to apply a saidalternating electrical current at a first frequency and concurrently tocontrol the voltage detector(s) to measure said first and secondvoltages, and to further control the electrical current unit to applysaid alternating electrical current at a second frequency exceeding thefirst frequency and concurrently to control the voltage detector(s) tomeasure said first and second voltages; a calculating unit arranged tocalculate a first electrical resistance value using the first and secondvoltages measured at said first frequency, and a second electricalresistance value and a reactance value using said first and secondvoltages measured at said second frequency; wherein the calculating unitis arranged to generate a value representing the relative volume of redblood cells in the liquid sample (haematocrit) according to the firstand second electrical resistance values and the electrical reactancevalue.

Paragraph 4: A sampling apparatus according to Paragraph 1 and Paragraph3 arranged to generate both said value representing the relative volumeof red blood cells in the liquid sample (haematocrit) and to generatesaid value representing the concentration of glycated haemoglobin(HbA1c) in a liquid sample using said haematocrit value.

Paragraph 5: A sampling apparatus according to any of Paragraphs 1 to 4including the sampling plate according to any of claims 1 to 14 in whicheach one of said two drive electrodes of the sampling plate is adaptedto electrically connect to a respective one of said two (or more)current output terminals concurrently, and in which each one of said twosensing electrodes is adapted to electrically connect to a respectiveone of the first voltage input terminal and the second voltage inputterminal concurrently, thereby to connect the two drive electrodes andthe two sensing electrodes to the sampling apparatus simultaneously forelectrical communication therewith.

Paragraph 6: A sampling apparatus according to any of Paragraphs 1 to 5comprising an integrated circuit arranged for measuring said firstvoltage and said second voltage, and responsive to the control unit toapply said alternating current accordingly.

Paragraph 7: A sampling apparatus according to any of Paragraphs 1 to 6in which the first frequency has a value in the range 1 KHz to 150 KHz.

Paragraph 8: A sampling apparatus according to any of Paragraph 1 to 7in which the second frequency has a value in the range 500 KHz to 1.5MHz.

Paragraph 9: A sampling apparatus according to any of Paragraph 1 to 8in which said direct (DC) voltage is substantially constant in value.

Paragraph 10: A sample measurement method for performing electricalmeasurements on a liquid sample containing blood, the method comprising:receiving the liquid sample on a sample plate comprising electrodeterminals which are separated by a spacing adapted to be bridged byblood from the liquid sample and which comprise a reagent to react withfree glucose in the liquid sample; and applying to the electrodes analternating electrical current having a given frequency to generate afirst alternating potential difference across the spacing between theelectrode terminals; applying between the electrode terminals asubstantially direct (DC) electrical potential difference of a givenmagnitude; determining a value of a first electrical reactance of theliquid sample bridging said spacing for said given frequency; removingthe substantially direct (DC) electrical potential difference frombetween the two electrode terminals; applying to the electrodes thealternating electrical current having said given frequency to generate asecond alternating potential difference across the spacing between theelectrode terminals; determining a value of a second electricalreactance of the liquid sample bridging said spacing for said givenfrequency; generating a value representing the concentration of glycatedhaemoglobin (HbA1c) in the blood within the sample according to thefirst electrical reactance value, the second electrical reactance valueand a value of the relative volume of red blood cells in the liquidsample (haematocrit).

Paragraph 11: A sample measurement method for performing electricalmeasurements on a liquid sample containing blood, the method comprising:receiving the liquid sample on a sample plate comprising electrodeterminals which are separated by a spacing adapted to be bridged byblood from the liquid sample; and applying to the electrodes analternating electrical current having a first signal frequency togenerate a first alternating potential difference across the spacingbetween the electrode terminals; determining a value of a firstelectrical resistance of the liquid sample bridging said spacing forsaid first signal frequency; applying to the electrodes an alternatingelectrical current having a second signal frequency exceeding said firstsignal frequency to generate a second alternating potential differenceacross the spacing between the electrode terminals; determining a valueof a second electrical resistance and a value of a reactance of theliquid sample bridging said spacing for said second signal frequency;generating a value for the relative volume of red blood cells(haematocrit) in the liquid sample according to the first electricalimpedance value and the second electrical impedance value.

Paragraph 12: A sample measurement method according to Paragraph 10including generating said value representing the relative volume of redblood cells in the liquid sample (haematocrit) according to Paragraph11.

Paragraph 13: A sample measurement method according to any of Paragraphs10 to 12 in which said direct (DC) voltage is substantially constant invalue.

The embodiments of the invention described above are intended to beillustrative of preferred implementations of the invention and variants,modifications and alterations to those implementations, such as would bereadily apparent to the skilled person, are encompassed within the scopeof the invention as defined e.g. by the claims.

TABLE 1 STRIP NO. R₁ R₂ X₃ MEASURED HCT ACTUAL HCT 1 318.2 232.3 5851.7049034 52 2 320.1 234.7 59 51.9084503 3 318.7 232.9 58 51.6905695 4320.2 233 58 51.751151 5 318.2 232.1 58 51.7171343 6 318.3 232.2 5851.7154794 7 318.4 232.2 58 51.7199399 8 317.5 230.5 57 51.5145174 9319.2 232.6 58 51.7311329 10 320.05 232.8 58 51.7566914 Strip No. R₁ R₂X₃ Measured HCT Actual HCT 1 240 190.3 35 42.7028225 42 2 240.3 190.5 3542.7056454 3 240.7 190.4 35 42.7367189 4 241 190.8 35.5 42.9444675 5240.7 190.5 35 42.7292629 6 240.9 190.9 35.5 42.9311337 7 240.7 190.8 3542.7069183 8 241 191 36 43.1463773 9 241.3 191.2 35 42.7125328 10 240190.2 35 42.7102863 1 195.3 165.8 18 31.4260775 31 2 193.1 163.8 1831.4375429 3 194.7 165.2 18 31.4338656 4 194.3 165.3 18 31.3960694 5193.1 163.7 18 31.4462147 6 193.1 163.8 18 31.4375429 7 193.1 163.7 1831.4462147 8 192.5 163.6 18 31.4107008 9 192.7 163.6 18 31.4254464 10192.8 163.5 18 31.4414958

The invention claimed is:
 1. A sampling plate comprising: a sample zonefor receiving a liquid sample; two drive electrodes with separaterespective electrode terminals spaced within the sample zone by aspacing for receiving said liquid sample within the sample zone for usein driving an electrical signal through the sample; two sensingelectrodes with separate respective electrode terminals spaced withinthe sample zone between the electrode terminals of the two driveelectrodes within the sample zone for use in sensing an electricalsignal generated by the drive electrodes within said sample.
 2. Asampling plate according to claim 1 in which the two sensing electrodeterminals present to each other opposing sides which define between theman elongate sensing gap extending along the sample zone for receivingsaid sample therein.
 3. A sampling plate according to claim 2 in whichthe width of the sensing gap is greater than about 90 microns and lessthan about 160 microns.
 4. A sampling plate according to claim 2 inwhich the sensing gap has a substantially uniform width along at least apart of its length.
 5. A sampling plate according to claim 2 in whichthe two drive electrode terminals present to each other opposing sideswhich define between them an elongate drive gap extending along thesample zone for receiving said sample therein whereby the driveelectrodes are adapted to drive electrical signal transversely acrossthe drive gap wherein the drive gap has a substantially uniform widthalong at least a part of its length.
 6. A sampling plate according toclaim 2 in which the two sensing electrode terminals present to eachother opposing sides which define between them an elongate sensing gapextending along the sample zone for receiving said sample therein, andin which the sensing gap and/or the drive gap has a substantiallyuniform width along at least a part of its length.
 7. A sampling plateaccording to claim 1 in which the two drive electrode terminals presentto each other opposing sides which define between them an elongate drivegap extending along the sample zone for receiving said sample thereinwhereby the drive electrodes are adapted to drive electrical signaltransversely across the drive gap.
 8. A sampling plate according toclaim 7 in which the sensing gap extends along the drive gap.
 9. Asampling plate according to claim 8 in which the two sensing electrodeterminals present to each other opposing sides which define between theman elongate sensing gap extending along the sample zone for receivingsaid sample therein, and in which the sensing gap and/or the drive gaphas a substantially uniform width along at least a part of its length.10. A sampling plate according to claim 1 in which a said driveelectrode terminal and an adjacent sensing electrode terminal present toeach other opposing sides which define between them an elongatepartitioning gap extending along the sample zone to define a partitionbetween the adjacent terminals within the sample zone.
 11. A samplingplate according to claim 10 in which the partitioning gap has asubstantially uniform width along at least a part of its length.
 12. Asampling plate according to claim 10 in which the width of thepartitioning gap is about 1.5 times the width of the sensing gap.
 13. Asampling plate according to claim 1 in which the sensing electrodeterminals are formed on a surface of the sampling plate within thesample zone.
 14. A sampling plate according to claim 13 in which thedrive electrode terminals are formed on a surface of the sampling platein common with the sensing electrode terminals within the sampling zone.15. A sampling plate according to claim 1 in which each of the driveelectrodes and each of the sensing electrodes is in electricalcommunication with respective electrical contact zones provided on thesampling plate which are exposed for electrical connectionsimultaneously with an external drive current source and externalsensing circuitry, respectively.
 16. A sampling plate according to claim1 in which the sample zone comprises a reagent to react with freeglucose in the liquid sample.
 17. A sampling plate according to claim 1comprising a plurality of said sample zones for receiving said liquidsample, each with two drive electrodes with separate respectiveelectrode terminals spaced by a spacing for receiving said liquid samplewithin each respective sample zone for use in driving an electricalsignal through the sample, and each with two sensing electrodes withseparate respective electrode terminals spaced between the electrodeterminals of the two drive electrodes for use in sensing an electricalsignal generated by the drive electrodes within said sample.